EP1931711B1 - Antibodies against 25-hydroxyvitamin d - Google Patents

Antibodies against 25-hydroxyvitamin d Download PDF

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Publication number
EP1931711B1
EP1931711B1 EP06805884A EP06805884A EP1931711B1 EP 1931711 B1 EP1931711 B1 EP 1931711B1 EP 06805884 A EP06805884 A EP 06805884A EP 06805884 A EP06805884 A EP 06805884A EP 1931711 B1 EP1931711 B1 EP 1931711B1
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Prior art keywords
hydroxyvitamin
vitamin
antibodies
conjugate
test
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German (de)
English (en)
French (fr)
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EP1931711A1 (en
Inventor
Erasmus Huber
Juergen Becker
Werner Kraus
Apostolos Kyriatsoulis
Rudolf Vogel
Nicole Horn
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors

Definitions

  • the present invention concerns processes for the production of antibodies against 25-hydroxyvitamin D, the antibodies produced according to the invention as well as methods for detecting 25-hydroxyvitamin D using these antibodies.
  • vitamin D An adequate supply of vitamin D is vital as the term "vitamin" already suggests.
  • a deficiency of vitamin D leads to severe diseases such as rickets or osteoporosis.
  • vitamin D was still regarded as a single substance at the beginning of the last century, the vitamin D system has developed further in the course of the last three decades into a complex and manifold network of vitamin D metabolites.
  • more than 40 different vitamin D metabolic products are known ( Zerwekh, J.E., Ann. Clin. Biochem. 41 (2004) 272-281 ).
  • Vitamin D 3 that is produced in the skin binds to the so-called vitamin D-binding protein which transports it into the liver where it is converted into 25-hydroxyvitamin D 3 by 25-hydroxylation.
  • a multitude of other tissues are nowadays known to be involved in vitamin D metabolism in addition to the skin and liver, the two organs that have already been mentioned (refer to Schmidt-Gayk, H. et al. (eds.), "Calcium regulating hormones, vitamin D metabolites and cyclic AMP", Springer Verlag, Heidelberg (1990), pp. 24-47 ).
  • 25-Hydroxyvitamin D and more specifically 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3 are the central storage forms of vitamin-D in the human organism with regard to their amounts. When needed these precursors can be converted in the kidneys to form the biologically active 1 ⁇ ,25-dihydroacyvitamin D, the so-called D hormone.
  • the biologically active vitamin D regulates among others calcium uptake from the intestine, bone mineralization and it influences a large number of other metabolic pathways such as e.g. the insulin system.
  • vitamin D is of little benefit when determining the vitamin D status of a patient, because concentrations of vitamin D (vitamin D 2 and vitamin D 3 ) fluctuate greatly depending on food uptake.
  • vitamin D has a relatively short biological half-life in the circulation (24 hours) and it is therefore also for this reason not a suitable parameter for determining the vitamin D status of a patient.
  • physiologically active forms of vitamin D (1,25-dihydroxyvitamin D). These biologically active forms also occur in relatively small and highly fluctuating concentrations compared to 25-hydroxyvitamin D. For all these reasons the quantification of 25-hydroxyvitamin D in particular is a suitable means to globally analyse the total vitamin D status of a patient.
  • Armbruster, F.P. et al. (WO 99/67211 ) teach that a serum or plasma sample should be prepared for vitamin D determination by ethanol precipitation. In this method the protein precipitate is removed by centrifugation and the ethanolic supernatant contains soluble vitamin D metabolites. These can be measured in a competitive binding assay.
  • EP 0 753 743 teaches that the proteins can be separated from blood or serum samples using a periodate salt.
  • vitamin D compounds are determined in the protein-free supernatant from the samples treated with periodate.
  • acetonitrile is recommended for the extraction of serum or plasma samples (e.g. in the radioimmunoassay from DiaSorin or in the vitamin D test from the "Immundiagnostik” Company).
  • EP 03 120 360 and Kobayashi et al disclose the preparation of 25-hydroxy vitamin D3 derivatives bearing a hapten-carrier in position 3.
  • Hummer et al (Scand. J. clin. Lab. Invest., 1984, vol. 44, pages 163-167 ) describes a method for determining 25OHD3 in serum by adding vitamin D2 in the sample.
  • the present invention concerns a process for producing antibodies against 25-hydroxyvitamin D which comprises the following steps:
  • the invention concerns antibodies against 25-hydroxyvitamin D 3 which have a cross-reaction with 25-hydroxyvitamin D 2 of the order of magnitude of 10 % to 1000%.
  • the present application also describes how the antibodies according to the present invention can be used for an automated test to detect 25-hydroxyvitamin D.
  • test kit for detecting 25-hydroxyvitamin D which contains the reagent compositions required for the test procedure and among others the antibodies against 25-hydroxyvitamin D according to the invention.
  • the present invention concerns a process for producing antibodies against 25-hydroxyvitamin D which comprises the following steps:
  • vitamin D is understood to include the forms of vitamin D 2 and vitamin D 3 according to the following structural formulae I and II.
  • the 25-hydroxyvitamin D denotes vitamin D metabolites that are hydroxylated at position 25 of the structural formulae I and II i.e. 25-hydroxyvitamin D 2 as well as 25-hydroxyvitamin D 3 .
  • 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3 are particularly relevant forms of vitamin D for diagnostics.
  • 1,25-Dihydroxyvitamin D refers to the active forms of vitamin D (the so-called D hormones) that have a hydroxylation at position 1 as well as at position 25 of the structural formulae I and II.
  • vitamin D metabolites are 24-dihydroxyvitamin D 2 and 25-dihydroxyvitamin D 2 as well as 24-dihydroxyvitamin D 3 and 25-dihydroxyvitamin D 3 .
  • hapten is understood by a person skilled in the art as a substance which per se is not immunogenic but, by coupling to a larger carrier molecule, is present in a form against which antibodies can be generated.
  • Suitable carrier materials for the production of hapten conjugates are known to a person skilled in the art. Bovine serum albumin, ⁇ -galactosidase or the so-called keyhole limpet hemocyanine (KLH) are usually used as carrier materials.
  • KLH has proven to be a particularly suitable carrier for the method according to the invention. Hence a conjugate of 25-hydroxyvitamin D and KLH is preferably used for the immunization.
  • Various positions of the structures as they are shown in formula I and II are in : principle suitable for activation and coupling to a carrier material. Coupling via position 3 of 25-hydroxyvitamin D 2 or 25-hydroxyvitamin D 3 has for example proven to be favourable for the generation of antibodies which bind a 25-hydroxyvitamin D in a suitable manner.
  • a conjugate is used in an immunization method according to the invention which contains 25-hydroxyvitamin D 3 or 25-hydroxyvitamin D 2 that has been coupled via position 3 of the backbone (cf. formulae I and II).
  • 25-hydroxyvitamin D 3 is complementary to 25-hydroxyvitamin D 2 and conversely 25-hydroxyvitamin D 2 is complementary to 25-hydroxyvitamin D 3 .
  • the same position of the vitamin D backbone for chemical coupling in the 25-hydroxyvitamin D conjugate used for the immunization and in the matrix used for the immunosorption.
  • the coupling in the 25-hydroxyvitamin D 3 conjugate is preferably via position 3 of 25-hydroxyvitamin D 3 for the immunization and 25-hydroxyvitamin D 2 is also preferably coupled to the matrix at position 3.
  • the converse procedure is also successful i.e. immunization with a 25-hydroxyvitamin D 2 conjugate and immunosorption with a matrix to which 25-hydroxyvitamin D 3 is coupled.
  • a 25-hydroxyvitamin D 2 conjugate is used as the immunogen conjugate and the antibodies generated with this immunogen are immuno-adsorbed onto a 25-hydroxyvitamin D 3 matrix.
  • EAH-Sepharose has proven to be particularly suitable as the matrix material for the immunosorption.
  • the antibodies contained in the serum or plasma from an immunization against 25-hydroxyvitamin D 3 or 25-hydroxy-vitamin D 2 are purified by immunosorption using a matrix which contains 25-hydroxyvitamin D 2 or 25-hydroxyvitamin D 3 .
  • EAH-Sepharose is a preferred column material.
  • the present invention concerns for example antibodies against 25-hydroxyvitamin D 3 which have a cross-reaction of 10 % to 1000 % with 25-hydroxyvitamin D 2 .
  • the cross-reaction with the complementary 25-hydroxyvitamin D form is also preferably in a range of 20 % to 500 %.
  • the extent of cross-reactions is determined in an immunological test method using the antibodies produced according to the present invention.
  • An antibody produced against 25-hydroxyvitamin D 3 as a hapten for example has a cross-reaction of 10 % for 25-hydroxyvitamin D 2 if, when using the same analyte concentration of 25-hydroxyvitamin D 2 or 25-hydroxyvitamin D 3 , only a tenth of 25-hydroxyvitamin D 3 is read-off on a calibration curve generated with 25-hydroxyvitamin D 3 .
  • the antibodies against 25-hydroxyvitamin D produced by a process according to the invention have proven to be suitable for use in an automated test for 25-hydroxyvitamin D.
  • the present invention preferably concerns the use of an antibody against 25-hydroxyvitamin D in an immunological test for the detection of 25-hydroxyvitamin D.
  • the test for 25-hydroxyvitamin D is preferably completely automated.
  • the antibodies according to the invention are particularly preferably used in a test that can be carried out on automated Elecsys ® analyzers from Roche Diagnostics.
  • test kit which contains all components required for the detection of 25-hydroxyvitamin D.
  • a preferred test kit for detecting 25-hydroxyvitamin D is in particular characterized in that such a kit contains an antibody against 25-hydroxy-vitamin D which recognizes both forms of 25-hydroxyvitamin D i.e. has a cross-reaction of 10 % to 1000 % to the complementary form of 25-hydroxyvitamin D in each case.
  • the test is preferably carried out as a competitive immunoassay in which the antibodies against 25-hydroxyvitamin D according to the invention are preferably used as a detection reagent.
  • a 25-hydroxyvitamin D "wall antigen" added in a defined amount to the test competes with the 25-hydroxyvitamin D from the sample for the binding sites of the detection antibody. The more 25-hydroxyvitamin D is present in the sample the smaller is the detection signal.
  • the form of 25-hydroxyvitamin D present as the wall antigen in the competitive test corresponds to the form that is used in the immunosorption. If one for example immunizes with an immunogen containing 25-hydroxyvitamin D 3 , immunosorption is carried out on a 25-hydroxy-vitamin D 2 matrix and a 25-hydroxyvitamin D 2 derivative is preferably used in the test as the wall antigen.
  • the wall antigen is preferably also modified at the same ring position as the immunogen and as the 25-hydroxyvitamin D used on the matrix for immunosorption.
  • the present invention concerns an immunological detection method for 25-hydroxyvitamin D in which a polyclonal antibody is used which was obtained by immunization with a 25-hydroxyvitamin D conjugate and immunosorption to the complementary 25-hydroxyvitamin D conjugate and wherein in a competitive test a derivative of the 25-hydroxyvitamin D complementary to the immunogen is used as the wall antigen.
  • 25-hydroxyvitamin D 3 was chemically activated at position 3 (cf formula II) and coupled to KLH as an immunogen support.
  • This synthesis via the intermediate steps 25-hydroxyvitamin D 3 -3-hemisuccinate and 25-hydroxyvitamin D 3 -3-hemisuccinate-N-hydroxysuccinimide ester is shown schematically in figure 1 .
  • the antibodies are produced in sheep.
  • the 25-hydroxyvitamin D 3 -3-hemisuccinate KLH conjugate from example 1 is used for the immunization.
  • the immunization dosage is 0.1 mg per animal.
  • the first immunization is carried out in complete Freud's adjuvant. Further immunizations take place at .4 week intervals in incomplete Freud's adjuvant over a period of 10 months. Serum is collected in the middle of each immunization interval.
  • An immunadsorber which contains conjugated 25-hydroxyvitamin D 2 as the specificity determinant is prepared for the immunochromatographic purification of the polyclonal antibodies.
  • the immunadsorber is obtained by the following steps:
  • 25-hydroxyvitamin D 2 (Fluka No. 17937) is dissolved in a 25 ml three necked round bottom flask with an internal thermometer in 10 ml dry acetonitrile under an argon atmosphere. 1.5 ml tert.-butanol/acetonitrile (9:1) is added to the solution and cooled to 6°C in an ice bath. Subsequently 820 ⁇ l of an acrylonitrile solution (86 ⁇ l acrylonitrile in 1.0 ml acetonitrile) is added and stirred for 15 minutes at 6°C.
  • an acrylonitrile solution 86 ⁇ l acrylonitrile in 1.0 ml acetonitrile
  • reaction solution is diluted with 10 ml methyl-tert.-butyl ether and washed twice with 10 ml H 2 O each time.
  • the organic phase is dried with about 1 g anhydrous sodium sulfate, filtered over a G3 glass frit and evaporated on a rotary evaporator. It is dried in a high vacuum to a viscous clear residue with a mass of about 55 mg.
  • the entire nitrile obtained above is dissolved in 15 ml diethyl ether and admixed with a suspension of 7.5 mg lithium hydride in 7.5 ml diethyl ether while stirring.
  • the reaction mixture is stirred for 1 hour at room temperature. Afterwards a suspension of 38.4 lithium aluminium hydride in 6.6 ml diethyl ether is added. This results in a strong turbidity of the mixture.
  • the reaction mixture is stirred for a further hour at room temperature, then the reaction mixture is cooled to 0-5°C in an ice bath and 35 ml water is carefully added.
  • the pH is made strongly basic by addition of 6.6 ml 10 M potassium hydroxide solution.
  • eluant A Millipore H2O + 0.1 % trifluoroacetic acid
  • eluant B 95 % acetonitrile + 5 % Millipore H2O + 0.1 % TFA
  • EAH Sepharose (Amersham Biosciences, No. 17-0569-03) is washed with 200 ml 0.5 M sodium chloride solution on a G3 glass frit and equilibrated with 200 ml 0.03 M potassium phosphate buffer pH 7.1. After excess liquid has drained off through the frit, the suspension is taken up in 200 ml of the same buffer and 1.7 mg (2.3 ⁇ mol) of the N-hydroxysuccinimide ester in 10 ml DMSO is added. The reaction mixture is agitated overnight at room temperature on a shaker.
  • the lyophilisate is dissolved in PBS, aggregates are removed by chromatography on Superdex 200 ® and the immunoadsorbed polyclonal antibodies obtained in this manner are used in a further step.
  • the immunoaffinity matrix is regenerated with 1 M propionic acid and preserved in a solution of PBS containing 0.9 % sodium azide.
  • the 25-hydroxyvitamin D determinations are carried out by means of HPLC (test for 25(OH)vitamin D 3 , from the "Immundiagnostik” Company, Bensheim, order No. KC 3400) or by means of LC-MS-MS ( Vogeser, M. et al., Clin. Chem. 50 (2004) 1415-1417 ) as described in the literature.
  • the affinity-purified antibodies according to example 2.3 e) are transferred to 100 mM potassium phosphate buffer, pH 8.5 and the protein concentration is adjusted to 1 mg/ml.
  • the ruthenylation reagent ruthenium (II) tris (bipyridyl)-N-hydroxysuccinimide ester) is dissolved in DMSO and added to the antibody solution at a molar ratio of 7.5 to 1. After a reaction time of 60 min the reaction is stopped by addition of 1-lysine and the excess labelling reagent is separated by gel permeation chromatography on Sephadex G25.
  • the sample is measured using an Elecsys ® system from the Roche Diagnostics company. 25 ⁇ l sample is mixed with 30 ⁇ l release reagent and simultaneously or sequentially with 15 ⁇ l ruthenylated detection antibody and incubated for 9 minutes. In the next step the biotinylated wall antigen (50 ⁇ l) is added and the pH value is kept in the desired range by further addition of release reagent (50 ⁇ l). After a further 9 minutes incubation magnetizable polystyrene particles coated with streptavidin (SA) (30 ⁇ l) are added and after a further incubation for 9 minutes, the amount of bound ruthenylated antibody is determined as usual.
  • SA streptavidin
  • the solution containing the ruthenylated ⁇ 25-OH-vitamin D> antibody conjugate contains: 20 mM phosphate buffer, pH 6.5 0.1 % oxypyrion 0.1 % MIT (N-methylisothiazolone-HCl) 10 % DMSO (dimethyl sulfoxide) 1 % EtOH (ethanol) 0.1 % polydocanol 1 % rabbit IgG (DET) 2.0 ⁇ g/ml PAB-Ru (from example 3.2)
  • the release reagent contains: 220 mM acetate buffer, pH 4.0 0.1 % oxypyrion 0.1 % MIT 10 % DMSO 1% EtOH 0.1 % polydocanol 0.2 % rabbit IgG
  • the solution with the biotinylated wall antigen contains: 20 mM phosphate buffer, pH 6.5 0.1 % oxypyrion 10 % DMSO 1% EtOH 0.1% polydocanol 0.2 % rabbit IgG 0.18 ⁇ g/ml Ag-Bi (from example 3.1)
  • the suspension with SA-coated latex particles contains: 0.72 mg/ml SA-coated magnetizable polystyrene particles having a binding capacity of 470 ng/ml.

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EP06805884A 2005-09-29 2006-09-27 Antibodies against 25-hydroxyvitamin d Revoked EP1931711B1 (en)

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EP05021247 2005-09-29
PCT/EP2006/009360 WO2007039193A1 (en) 2005-09-29 2006-09-27 Antibodies against 25-hydroxyvitamin d
EP06805884A EP1931711B1 (en) 2005-09-29 2006-09-27 Antibodies against 25-hydroxyvitamin d

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EP (1) EP1931711B1 (es)
JP (1) JP2009509992A (es)
CN (1) CN101273062A (es)
AT (1) ATE427965T1 (es)
CA (1) CA2624124A1 (es)
DE (1) DE602006006200D1 (es)
ES (1) ES2323900T3 (es)
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US9616109B2 (en) 2014-10-22 2017-04-11 Extend Biosciences, Inc. Insulin vitamin D conjugates
US9789197B2 (en) 2014-10-22 2017-10-17 Extend Biosciences, Inc. RNAi vitamin D conjugates
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US10222389B2 (en) 2013-09-17 2019-03-05 Biomerieux Solution for dissociating vitamin D from vitamin D-binding protein, associated detection method and use
US10220047B2 (en) 2014-08-07 2019-03-05 Opko Ireland Global Holdings, Ltd. Adjunctive therapy with 25-hydroxyvitamin D and articles therefor
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EP0873126A4 (en) * 1995-12-29 2000-04-19 A And D Assay Inc BRANDED VITAMIN COMPOUNDS AND THEIR USE
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US9173950B2 (en) 2012-05-17 2015-11-03 Extend Biosciences, Inc. Vitamin D-ghrelin conjugates
US9289507B2 (en) 2012-05-17 2016-03-22 Extend Biosciences, Inc. Carriers for improved drug delivery
US9884124B2 (en) 2012-05-17 2018-02-06 Extend Biosciences, Inc. Carriers for improved drug delivery
US10197581B2 (en) 2013-03-14 2019-02-05 Enzo Life Sciences, Inc. Vitamin D assays
US9585934B2 (en) 2014-10-22 2017-03-07 Extend Biosciences, Inc. Therapeutic vitamin D conjugates
US9616109B2 (en) 2014-10-22 2017-04-11 Extend Biosciences, Inc. Insulin vitamin D conjugates
US9789197B2 (en) 2014-10-22 2017-10-17 Extend Biosciences, Inc. RNAi vitamin D conjugates
US10702574B2 (en) 2014-10-22 2020-07-07 Extend Biosciences, Inc. Therapeutic vitamin D conjugates
US11116816B2 (en) 2014-10-22 2021-09-14 Extend Biosciences, Inc. Therapeutic vitamin d conjugates
US12076366B2 (en) 2014-10-22 2024-09-03 Extend Biosciences, Inc. Therapeutic vitamin D conjugates

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ATE427965T1 (de) 2009-04-15
DE602006006200D1 (de) 2009-05-20
JP2009509992A (ja) 2009-03-12
EP1931711A1 (en) 2008-06-18
US20080317764A1 (en) 2008-12-25
ES2323900T3 (es) 2009-07-27
CN101273062A (zh) 2008-09-24
CA2624124A1 (en) 2007-04-12

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